This invention relates to antisense oligomers for use in treating a picornavirus, calicivirus, togavirus or flavivirus infection, antiviral treatment methods employing the oligomers, and methods for monitoring binding of antisense oligomers to a viral genome target site.
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RNA viruses cause many diseases in wildlife, domestic animals and humans. These viruses are genetically and antigenically diverse, exhibiting broad tissue tropisms and a wide pathogenic potential. The incubation periods of some of the most pathogenic viruses, e.g. the caliciviruses, are very short. Viral replication and expression of virulence factors may overwhelm early defense mechanisms (Xu, 1991) and cause acute and severe symptoms.
There are no specific treatment regimes for many viral infections. The infection may be serotype specific and natural immunity is often brief or absent (Murray et al., 1998). Immunization against these virulent viruses is impractical because of the diverse serotypes. RNA virus replicative processes lack effective genetic repair mechanisms, and current estimates of RNA virus replicative error rates are such that each genomic replication can be expected to produce one to ten errors, thus generating a high number of variants (Hollan, 1993). Often, the serotypes show no cross protection, such that infection with any one serotype does not protect against infection with another. For example, vaccines against the vesivirus genus of the caliciviruses would have to provide protection against over 40 different neutralizing serotypes (Smith et al., 1998a), and vaccines for the other genera of the Caliciviridae are expected to have the same limitations.
Antisense agents have been proposed for treating various types of viral infection. In general, the specific proposals to date can be classified according to the type of virus targeted, the viral-genome target, and the type of oligonucleotide backbone employed in the antisense compound. Among the viruses that have been targeted are vesicular stomatitis virus (Robbins and Lebleu, 1999), influenza virus (Mizuta et al., 1999), hepatitis B virus (Wu and Wu, 1992), human papilloma virus (Alvarez-Salas et al., 1999), herpes simplex virus (Aurelian and Smith, 2000), HIV (Kusunoki et al., Wei et al., 2000) and foot-and-mouth disease virus (Gutierrez et al., 1993). Viral genome targets that have been proposed include the IE-2 gene of cytomegalovirus (Green et al., 2000), a stem-loop structure at the 5xe2x80x2 non-coding region, the translation initiation codon, a core protein coding sequence of the hepatitis C virus, and the second functional initiator AUG of the foot-and-mouth disease virus (Hanecak et al., 1996; Alt et al., 1995; Gutierrez et al., 1993). Finally, a wide variety of antisense backbone structures have been proposed, including the negatively charged phosphorothioate (PSO) backbone oligomers, particularly the phosphorothioate oligodeoxynucleotides (Hanecak et al., 1996; Alt et al., 1995; Gutierrez et al., 1993) and uniformly modified 2xe2x80x2-methoxyethoxy phosphodiester oligonucleotide (Hanecak et al., 1996).
Discovery and development generally involves demonstration of antiviral activity in cell culture. A compilation of antiviral experiments in cell culture is provided in Table 1 below.
Clinical trials have been initiated for antisense therapeutics targeting HIV, HPV, CMV and HCV (Table 2 below), all using phosphorothioate-linked oligonucleotides. As seen, the clinical trial experience to date indicates some failures, although antisense against CMV infection (ISIS2922) has been approved by the FDA, making this the only antisense agent approved by the FDA to date.
The initial optimism towards antisense approaches to effective antiviral therapeutics has been blunted. Many of the effective antisense strategies employed in cell culture models (e.g. those in Table 1) have not successfully proceeded to clinical trials. The slow progress is due in part to the lack of robust cell culture models. For example, the HIV isolates that infect cultured cells do not generally reflect those found in the infected population, and the cell culture models do not integrate the roles of the multiple cell types infected. This problem is compounded by the lack of appropriate pre-clinical animal models for the fill exploitation of viral gene expression and replication in vivo. Again, in the case of HIV, the human virus either does not infect animals, or, when primates are infected, they do not develop pathology similar to that seen in humans. The risk in developing antisense antiviral agents without robust culture models and appropriate animal models is great.
Thus, there remains a need for an effective antiviral therapy in several virus families, including small, single-stranded, positive-sense RNA viruses in the picornavirus, calicivirus, togavirus and flavivirus families. To meet this need, an antisense agent must be substantially stable against nuclease degradation, able to be taken up readily by virus-infected host cells following compound administration, and targeted against an effective region of the viral genome, that is, able to shut down viral replication.
In one aspect, the invention provides an antiviral compound directed against an RNA virus from the picornavirus, calicivirus, togavirus or flavivirus families having a single-stranded, positive sense genome of less than 12 kb and a first open reading frame that encodes a polyprotein containing multiple functional proteins. The antiviral compound comprises a substantially uncharged oligomer having (a) a sequence of 12 to 40 subunits, supporting a targeting base sequence that is substantially complementary to a viral target sequence which spans the translation initiation region of said first open reading frame, and (b) a substantially uncharged backbone.
In a preferred embodiment, the oligomer is a morpholino oligomer, having a sequence or morpholino subunits. The subunits are generally connected by uncharged, phosphorus-containing intersubunit linkages, which joining the morpholino nitrogen of one subunit to the 5xe2x80x2 exocyclic carbon of an adjacent subunit. In one embodiment, these linkages are phosphorodiamidate linkages. For example, one embodiment of a morpholino subunit and phosphorodiamidate linkage may be represented by the structure: 
where Y1=O, Z=O, Pj is a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide (where base-pairing moieties on different subunits may be the same or different), and X is alkyl, alkoxy, thioalkoxy, or alkyl amino. In one embodiment, X=NR2, where each R is independently hydrogen or methyl. In an oligomer of such structural units, the phosphorus atom of one is bonded to the morpholino nitrogen of the next.
The substantially uncharged oligomer will typically have a Tm, with respect to binding to the viral target sequence, of greater than about 50xc2x0 C., as well as an ability to be actively taken up by mammalian cells. In addition, the compound can generally be recovered, in a heteroduplex form consisting of the oligomer and a complementary portion of the viral genome of the RNA virus, from the serum or urine of a mammalian subject, several hours after being administered to the subject.
In various embodiments, the antiviral compounds are directed against specific viruses or families. For example, selected embodiments include antiviral compounds directed against a picornavirus. Exemplary compounds include those having a targeting sequence having at least 90% homology to a sequence selected from the group consisting of:
(i) SEQ ID NO. 16, for a polio virus of the Mahoney and Sabin strains,
(ii) SEQ ID NO. 17, for a hepatitis A virus,
(iii) SEQ ID NO. 18, for a rhinovirus 14,
(iv) SEQ ID NO. 19, for a rhinovirus 16,
(v) SEQ ID NO. 20, for a rhinovirus 1B,
(vi) SEQ ID NOs. 21 and 22, for an Aphthovirus, and
(vii) SEQ ID NOs 23, 24 and 25, for a coxsackie virus.
Other embodiments include antiviral compounds directed against a calicivirus. Exemplary compounds include those having a targeting sequence having at least 90% homology to a sequence selected from the group consisting of:
(i) SEQ ID NOs. 27, 28, and 29, for a serotype Pan-1 vesivirus,
(ii) SEQ ID NO. 30, for a porcine vesivirus,
(iii) SEQ ID NO. 31, for a Norwalk virus, and
(iv) SEQ ID NO. 32, for a feline vesivirus.
Other embodiments include antiviral compounds directed against a togavirus. For use in inhibition of hepatitis E virus, the compound comprises an oligomer having a targeting sequence having at least 90% homology to a sequence selected from the group consisting of SEQ ID NOs: 33 and 34. Still other embodiments include antiviral compounds directed against a togavirus. For use in inhibition of a hepatitis C flavivirus, the compound comprises an oligomer having a targeting sequence with at least 90% homology to SEQ ID NO. 35.
In more specific embodiments, the compounds have the exact targeting sequences shown, and/or comprise phosphorodiamidate-linked morpholino oligomers. For example, compounds directed against the serotype Pan-1 vesivirus may comprise a phosphorodiamidate-linked morpholino oligomer (PMO) having a targeting sequence selected from the group consisting of SEQ ID NOs. 27, 28, and 29. A compound directed against the feline vesivirus may comprise a PMO having the targeting sequence SEQ ID NO. 31.
In a related aspect, the invention provides a method of inhibiting replication of an RNA virus from the picomavirus, calicivirus, togavirus or flavivirus families, having a single-stranded, positive sense genome of less than 12 kb, and a first open reading frame that encodes a polyprotein containing multiple functional proteins. The method comprises exposing the virus, or, typically, a cell infected with the virus, to a substantially uncharged morpholino oligomer having (a) a sequence of 12 to 40 subunits, supporting a targeting base sequence that is substantially complementary to a viral target sequence which spans the translation initiation region of the first open reading frame, and (b) a substantially uncharged backbone. In one embodiment of the method, the oligomer is administered to a mammalian subject infected with the virus. Preferred embodiments of the antisense compounds, with respect to properties and structure, are as described above.
In a further aspect, the invention provides a method of confirming the presence of an effective interaction between a picornavirus, calicivirus, togavirus or flavivirus infecting a mammalian subject, and a substantially uncharged antisense oligomer targeted against the infecting virus. The method comprises:
(a) administering the oligomer to the subject,
(b) at a selected time after said administration, obtaining a sample of a body fluid from the subject; and
(c) assaying the sample for the presence of a nuclease-resistant heteroduplex comprising the antisense oligomer and a complementary portion of the viral target sequence. As above, the oligomer has a sequence of 12 to 40 subunits, supporting a targeting base sequence that is substantially complementary to a viral target sequence which spans the translation initiation region of the first open reading frame (ORF1) of the infecting virus. Preferably, the oligomer is a morpholino oligomer, and has uncharged, phosphorus-containing intersubunit linkages joining the morpholino nitrogen of one subunit to the 5xe2x80x2 exocyclic carbon of an adjacent subunit. In one embodiment, the linkages are phosphorodiamidate linkages.
This method can be used in determining the effectiveness of treating a picornavirus, calicivirus, togavirus or flavivirus infection by administering the oligomer, by carrying out the described steps of administering, obtaining a sample, and assaying for heteroduplex at periodic intervals throughout a treatment period.
In addition, the method can be used in determining the identity of an infecting picornavirus, calicivirus, togavirus or flavivirus. The family or genus of such a virus can be determined by:
(a) providing a plurality of antisense oligomers, each having a base sequence that is substantially complementary to a viral target sequence of a plurality of known viruses selected from picornaviruses, caliciviruses, togaviruses or flaviviruses, wherein each said viral target sequence is (i) common to a virus family or genus, and (ii) not found in humans;
(b) administering at least one oligomer of the plurality to the subject,
(c) at a selected time after said administering, obtaining a sample of a body fluid from the subject;
(d) assaying the sample for the presence of a nuclease-resistant heteroduplex comprising the antisense oligomer and a complementary portion of the viral target sequence, and
(e) identifying the family or genus of the infecting virus, based on the presence or absence of a heteroduplex comprising an administered antisense oligomer and a complementary portion of said viral target base sequence.
For identification of a specific infecting picornavirus, calicivirus, togavirus or flavivirus, the following further steps can be carried out:
(a) providing a second plurality of antisense oligomers, each having a base sequence that is substantially complementary to a viral target sequence of one of a plurality of known viruses from the family or genus identified in step (e) above, wherein each said viral target sequence is (i) specific to one of said known viruses, and (ii) not found in humans;
(b) administering at least one oligomer of the plurality to the subject,
(c) at a selected time after said administering, obtaining a sample of a body fluid from the subject;
(d) assaying the sample for the presence of a nuclease-resistant heteroduplex comprising the antisense oligomer and a complementary portion of the viral target sequence, and
(e) identifying the infecting virus, based on the presence or absence of a heteroduplex comprising an administered antisense oligomer and a complementary portion of said viral target base sequence.
These and other objects and features of the invention will be more fully appreciated when the following detailed description of the invention is read in conjunction with the accompanying figures.